ES2402808T3 - Methods of obtaining polymorphic forms of 3- (4-amino-1-oxo-1,3-dihydro-isoindol-2-yl) -piperidine-2,6-dione - Google Patents

Abstract

A procedure to prepare 3- (4-amino-1-oxo-1, A procedure to prepare 3- (4-amino-1-oxo-1,3-dihydro-isoindol-2-yl) -piperidine-2,6 -diona hemi3-dihydro-isoindol-2-yl) -piperidine-2,6-dione crystalline hemihydrate, comprising the steps of: crystalline shidrate, comprising the steps of: suspending 3- (4-amino-1-oxo- 1,3-dihydro-isoindol-2-iuspender 3- (4-amino-1-oxo-1,3-dihydro-isoindol-2-yl) -piperidine-2,6-dione in water at a temperature l) - piperidine-2,6-dione in water at a temperature of 70 ° C to 80 ° C for 6 to 24 hours; filter 3- (4-70 ° C to 80 ° C for 6 to 24 hours; filter 3- (4-amino-1-oxo-1,3-dihydro-isoindole-2-yl) -piperidine-amino-1-oxo-1 , 3-dihydro-isoindol-2-yl) -piperidine-2,6-dione; and dry 3- (4-amino-1-oxo-1,3-dihydro-is2,6-dione; and dry 3- (4-amino-1-oxo-1,3-dihydro-isoindole-2-yl) -piperidine-2,6-dione in vacuo.oindol-2-yl) -piperidine-2,6-dione in vacuo.

Description

Methods of obtaining polymorphic forms of 3- (4-amino-1-oxo-1,3-dihydro-isoindol-2-yl) -piperidine-2,6-dione.

This invention relates to processes for preparing polymorphic forms of 3- (4-amino-1-oxo-1,3-dihydroisoindol-2-yl) -piperidine-2,6-dione. Compositions comprising polymorphic forms, methods of obtaining polymorphic forms and methods of their use for the treatment of diseases and conditions that include, but are not limited to, inflammatory diseases, autoimmune diseases and cancer are also described.

Many compounds may exist in different crystalline forms, or polymorphs, that exhibit different physical, chemical and spectroscopic properties. For example, certain polymorphs of a compound may be more readily soluble in particular solvents, may flow more easily, or may compress more easily than others. See, eg, P. DiMartino, et al., J. Thermal. Anal., 48: 447-458 (1997). In the case of drugs, certain solid forms may be more readily bioavailable than others, while others may be more stable under certain biological, manufacturing and storage conditions. This is particularly important from a regulatory point of view, since drugs are approved by agencies such as the United States Food and Drug Administration only if they meet exact standards of purity and characterization. In fact, regulatory approval of a polymorph of a compound, which exhibits certain solubility and physicochemical properties (including spectroscopic), typically does not imply easy approval of other polymorphs of that same compound.

In pharmaceutical techniques it is known that polymorphic forms of a compound affect, for example, the solubility, stability, flowability, fractability and compressibility of the compound, as well as the safety and efficacy of the pharmaceutical products that comprise it. See, eg, Knapman, K. Modern Drug Discoveries, 2000, 53. Therefore, the discovery of new polymorphs of a drug can provide a variety of advantages.

U.S. patents Nos. 5,635,517 and 6,281,230, both of Muller et al., Describe 3- (4-amino-1-oxo-1,3-dihydroisoindol-2-yl) -piperidine-2,6-dione, which is useful in the treatment and prevention of a wide range of ailments, which include, but are not limited to, inflammatory diseases, autoimmune diseases and cancer. New polymorphic forms of 3- (4-amino-1-oxo-1,3-dihydro-isoindol-2-yl) -piperidine-2,6-dione can promote the development of formulations for the treatment of these chronic diseases, and They can provide numerous formulation, manufacturing and therapeutic benefits.

This invention provides methods of obtaining polymorphs of 3- (4-amino-1-oxo-1,3-dihydro-isoindol-2-yl) piperidine-2,6-dione as defined in the appended claims.

Specific aspects of the invention can be understood with reference to the attached figures:

FIGURE 1 provides a powder X-ray diffraction pattern (XRPD) representative of Form A;

FIGURE 2 provides a representative IR spectrum of Form A;

FIGURE 3 provides a representative Raman spectrum of Form A;

FIGURE 4 provides a representative thermogravimetric analysis (TGA) curve and a differential scanning calorimetry (DSC) thermogram representative of Form A;

FIGURE 5 provides a moisture sorption / desorption isotherm representative of Form A;

FIGURE 6 provides a representative XRPD pattern of Form B;

FIGURE 7 provides a representative IR spectrum of Form B;

FIGURE 8 provides a representative Raman spectrum of Form B;

FIGURE 9 provides a representative TGA curve and a representative DSC thermogram of Form B;

FIGURE 10 provides representative TG-IR results of Form B;

FIGURE 11 provides a moisture sorption / desorption isotherm representative of Form B;

FIGURE 12 provides a representative XRPD pattern of Form C;

FIGURE 13 provides a representative IR spectrum of Form C;

FIGURE 14 provides a representative Raman spectrum of Form C;

FIGURE 15 provides a representative TGA curve and a DSC thermogram representative of the Form

C; FIGURE 16 provides representative TG-IR results of Form C; FIGURE 17 provides a moisture sorption / desorption isotherm representative of Form C; FIGURE 18 provides a representative XRPD pattern of Form D; FIGURE 19 provides a representative IR spectrum of Form D; FIGURE 20 provides a representative Raman spectrum of Form D; FIGURE 21 provides a representative TGA curve and a DSC thermogram representative of the Form

D; FIGURE 22 provides a moisture sorption / desorption isotherm representative of Form D; FIGURE 23 provides a representative XRPD pattern of Form E; FIGURE 24 provides a representative TGA curve and a DSC thermogram representative of the Form

AND; FIGURE 25 provides a moisture sorption / desorption isotherm representative of Form E; FIGURE 26 provides a representative XRPD pattern of Form F; FIGURE 27 provides a representative thermogram of Form F; FIGURE 28 provides a representative XRPD pattern of Form G; FIGURE 29 provides a representative DSC thermogram of Form G; FIGURE 30 provides a representative XRPD pattern of Form H; FIGURE 31 provides a representative TGA curve and a DSC thermogram representative of the Form

H; FIGURE 32 provides a representative XRPD pattern of Form B; FIGURE 33 provides a representative XRPD pattern of Form B; FIGURE 34 provides a representative XRPD pattern of Form B; FIGURE 35 provides a representative XRPD pattern of Form E; FIGURE 36 provides a representative XRPD pattern of a mixture of polymorphs; FIGURE 37 provides a representative TGA curve of Form B; FIGURE 38 provides a representative TGA curve of Form B; FIGURE 39 provides a representative TGA curve of Form B; FIGURE 40 provides a representative TGA curve of Form E; FIGURE 41 provides a representative TGA curve of a mixture of polymorphs; FIGURE 42 provides a representative DSC thermogram of Form B; FIGURE 43 provides a representative DSC thermogram of Form B; FIGURE 44 provides a representative DSC thermogram of Form B; FIGURE 45 provides a representative DSC thermogram of Form E; FIGURE 46 provides a DSC thermogram representative of a mixture of polymorphs; FIGURE 47 provides a UV-Vis scan of a dissolution medium; FIGURE 48 provides a UV-Vis scan of 0.04 mg / ml of 3- (4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)

piperidine-2,6-dione in the dissolution medium;

FIGURE 49 provides a UV-Vis scan of 0.008 mg / ml of 3- (4-amino-1-oxo-1,3-dihydro-isoindol-2-yl) piperidine-2,6-dione in the dissolution medium ;

FIGURE 50 provides a calibration curve for 3- (4-amino-1-oxo-1,3-dihydro-isoindol-2-yl) -piperidine-2,6dione;

FIGURE 51 provides a solubility curve of Form A;

FIGURE 52 provides a solubility curve of Form B;

FIGURE 53 provides an intrinsic dissolution of Forms A, B and E; Y

FIGURE 54 provides an intrinsic solution of Forms A, B and E.

As used herein, and unless otherwise indicated, the terms "treat", "treating" and "treatment" refer to the relief of a disease or disorder and / or at least one of its accompanying symptoms.

As used herein, and unless otherwise indicated, the terms "prevent", "preventing" and "prevention" refer to the inhibition of a symptom of a disease or disorder or of the disease itself.

As used herein, and unless otherwise indicated, the terms "polymorph" and "polymorphic form" refer to solid crystalline forms of a compound or complex. Different polymorphs of the same compound may exhibit different physical, chemical and / or spectroscopic properties. Different physical properties include, but are not limited to, stability (e.g., heat or light), compressibility and density (important in product formulation and manufacturing), and dissolution rates (which may affect bioavailability. ). Differences in stability may be the result of changes in chemical reactivity (eg, differential oxidation, such that a dosage form fades more rapidly when it is comprised of a polymorph than when it is comprised of another polymorph) or mechanical characteristics (e.g., tablets crumble in storage, since a kinetically favored polymorph becomes a thermodynamically more stable polymorph) or both (e.g., tablets in a polymorph are more susceptible to breakage at high humidity). The different physical properties of polymorphs can affect their processing. For example, one polymorph could be more prone to form solvates or it could be more difficult to release impurities by filtration or washing than another, due to, for example, the shape or size distribution of its particles.

Polymorphs of a molecule can be obtained by several methods known in the art. Such methods include, but are not limited to, recrystallization in the molten state, cooling in the molten state, recrystallization in solvent, desolvation, rapid evaporation, rapid cooling, slow cooling, vapor diffusion and sublimation. Polymorphs can be detected, identified, classified and characterized using well known techniques, such as, but not limited to, differential scanning calorimetry (DSC), thermogravimetry (TGA), powder X-ray diffractometry (XRPD), ray diffractometry Single crystal X, vibrational spectroscopy, dissolution calorimetry, solid-state nuclear magnetic resonance (NMR), infrared (IR) spectroscopy, Raman spectroscopy, hot-plate optical microscopy, scanning electron microscopy (SEM), electronic crystallography and analysis quantitative, particle size analysis (PSA), surface area analysis, solubility and dissolution rate.

As used herein to refer to the spectra or data presented in the form

graph (eg, XRPD, IR, Raman and NMR spectra), and unless otherwise indicated, the term "peak" is

refers to a peak or other special feature that one skilled in the art would recognize as not attributable to background noise.

The term "significant peaks" refers to peaks of at least the median size (eg, height) of other peaks in

the spectrum or data, or at least 1.5, 2 or 2.5 times the median size of other peaks in the spectrum or data.

As used herein, and unless otherwise indicated, the term "substantially pure", when used to describe a polymorph of a compound, means a solid form of the compound comprising that polymorph and is substantially free of other polymorphs of the compound. A representative substantially pure polymorph comprises more than 80% by weight of a polymorphic form of the compound and less than 20% by weight of other polymorphic forms of the compound, more preferably more than 90% by weight of a polymorphic form of the compound and less than 10 % by weight of other polymorphic forms of the compound, even more preferably more than 95% by weight of a polymorphic form of the compound and less than 5% by weight of other polymorphic forms of the compound, and most preferably more than 97% by weight of a polymorphic form of the compound and less than 3% by weight of other polymorphic forms of the compound.

Polymorphic forms

This invention is directed to processes for preparing polymorphic forms of 3- (4-amino-1-oxo-1,3-dihydroisoindol-2-yl) -piperidine-2,6-dione, which has the structure shown below:

This compound can be prepared according to the methods described in US Pat. Nos. 6,281,230 and

5,635,517. For example, the compound can be prepared by catalytic hydrogenation of 3- (4-nitro-1-oxo-1,3-dihydro-isoindol-2-yl) -piperidine-2,6-dione. 3- (4-Nitro-1-oxo-1,3-dihydro-isoindol-2-yl) -piperidine-2,6-dione can be obtained by reacting 2,6-dioxopieridin-3-ammonium chloride with 2 -methyl bromomethyl-4-nitrobenzoate in dimethylformamide in the presence of triethylamine. Methyl 2-bromomethyl-4-nitrobenzoate, in turn, is obtained from the corresponding methyl ester of nitro-ortho-toluic acid by conventional bromination with Nbromosuccinimide under the influence of light.

Polymorphs of 3- (4-amino-1-oxo-1,3-dihydro-isoindol-2-yl) -piperidine-2,6-dione can be obtained by techniques known in the art, including solvent recrystallization, desolvation, rapid evaporation, slow evaporation, fast cooling and slow cooling. Polymorphs can be prepared by dissolving a heavy amount of 3- (4-amino-1-oxo-1,3-dihydro-isoindol-2-yl) -piperidine-2,6-dione in various solvents at elevated temperatures. Then, the solutions of the compound can be filtered and allowed to evaporate, either in an open vial (for rapid hot evaporation) or in a vial covered with aluminum foil containing small holes (slow hot evaporation). Polymorphs can also be obtained from suspensions. Polymorphs can be crystallized from solutions or suspensions using various methods. For example, a solution created at an elevated temperature (eg, 60 ° C) can be filtered rapidly, and then allowed to cool to room temperature. Once at room temperature, the sample that did not crystallize can be transferred to a refrigerator, and then filtered. Alternatively, the solutions can be cooled rapidly by dissolving the solid in a solvent at an increased temperature (eg, 45-65 ° C) followed by cooling in a dry ice / solvent bath.

Form A is a non-solvated crystalline material, which can be obtained from non-aqueous solvent systems. Form B is a crystalline material, hemihydrate, which can be obtained from various solvent systems. Form C is a hemisolvated crystalline material that can be obtained from solvents such as, but not limited to, acetone. Form D is a solvated, crystalline polymorph, prepared from a mixture of acetonitrile and water. Form E is a crystalline material, dihydrated. Form F is a non-solvated crystalline material, which can be obtained from the dehydration of Form E. Form G is a non-solvated crystalline material, which can be obtained by suspending Forms B and E in a solvent such as, but not limited to, tetrahydrofuran (THF). Form H is a partially hydrated crystalline material that can be obtained by exposing Form E to 0% relative humidity. Each of these forms is discussed in detail below.

Also described is a composition comprising 3- (4-amino-1-oxo-1,3-dihydro-isoindol-2-yl) -piperidine-2,6-dione amorphous and 3- (4-amino-1-oxo-1 , Crystalline 3-dihydro-isoindol-2-yl) -piperidine-2,6-dione of the form A, BC, D, E, F, G or H. Specific compositions may comprise more than 50, 75, 90 or 95 weight percent of crystalline 3- (4-amino-1oxo-1,3-dihydro-isoindol-2-yl) -piperidine-2,6-dione.

A composition comprising at least two crystalline forms of 3- (4-amino-1-oxo-1,3-dihydro-isoindol-2-yl) -piperidine-2,6-dione (eg, a mixture) is also described. of polymorphic forms B and E).

Form A

The data described herein for Form A, as well as for Forms BH, were obtained using the experimental methods described in the Examples dealing with the measurements of XRPD, thermal analysis, spectroscopy, moisture sorption / desorption and NMR, provided later.

Form A can be obtained from various solvents, which include, but are not limited to, 1-butanol, butyl acetate, ethanol, ethyl acetate, methanol, methyl ethyl ketone and THF. Figure 1 shows a representative XRPD pattern of Form A. The pattern is characterized by peaks, preferably significant peaks, at 8, 14.5, 16, 17.5, 20.5, 24 and 26 degrees 2θ. Data from the representative IR and Raman spectra are provided in Figures 2 and 3.

The representative thermal characteristics of Form A are shown in Figure 4. The TGA data shows a small weight gain up to approximately 150 ° C, indicating a non-solvated material. Weight loss above 150 ° C is attributed to decomposition. The DSC curve of Form A exhibits an endotherm at 270 ° C.

Representative sorption and moisture desorption data are plotted in Figure 5. Form A does not exhibit a significant weight gain of 5 to 95% relative humidity. The equilibrium can be obtained at each stage of relative humidity. As the form dries from 95% back to 5% relative humidity, it tends to maintain its weight, such that at 5% relative humidity it has typically lost only 0.003% by weight from the beginning to the end. Form A is able to remain as a crystalline solid for 11 days when stored at 22, 45, 58 and 84% relative humidity.

Interconversion studies show that Form A can be converted to Form B in aqueous solvent systems and can be converted to Form C into solvent systems in acetone. Form A tends to be stable in anhydrous solvent systems. In aqueous systems and in the presence of Form E, Form A tends to become Form E.

When stored for a period of 85 days under two different temperature / relative humidity conditions (ambient temperature / 0% relative humidity (RH) and 40 ° C / 93% RH), typically the Form A does not become a different form.

In sum, Form A is a crystalline solid, not solvated, that melts at 270 ° C. Form A is weakly or non-hygroscopic, and appears to be the most thermodynamically stable anhydrous polymorph of 3- (4-amino-1-oxo-1,3-dihydroisoindol-2-yl) -piperidine-2,6-dione discovered until now.

Form B

Form B can be obtained from many solvents, which include, but are not limited to, hexane, toluene and water. Figure 6 shows a representative XRPD pattern of Form B, characterized by peaks at 16, 18, 22 and

27 degrees 2θ.

Proton NMR in solution confirms that Form B is a form of 3- (4-amino-1-oxo-1,3-dihydro-isoindol2-yl) -piperidine-2,6-dione. Representative IR and Raman spectra are shown in Figures 7 and 8, respectively. Compared to Form A, the IR spectrum for Form B has peaks at 3513 and 1960 cm-1.

Representative DSC and TGA data are shown for Form B in Figure 9. The DSC curve exhibits endotherms at 146 and 268 ° C. These events are identified as dehydration and fusion by hot plate microscopy experiments. Form B typically loses 3.1% of volatiles up to 175 ° C (by 0.46 moles of water). Comparison of the IR spectrum of volatiles with that of water indicates that they are water (see Figure 10). Calculations from TGA data indicate that Form B is a hemihydrate. The Karl Fischer water analysis also supports this conclusion.

Representative sorption and moisture desorption data are shown in Figure 11. Form B typically does not exhibit a significant weight gain of 5% to 95% relative humidity, when equilibrium is obtained at each stage of relative humidity. As Form B dries from 95% back to 5% relative humidity, it tends to maintain its weight, such that at 5% relative humidity it has typically gained only 0.022% by weight (0.03 mg) since the Start to the end. Form B does not convert into a different form after exposure to 84% relative humidity for ten days.

Interconversion studies show that Form B is typically converted into Form A in a THF solvent system, and typically converted to Form C into an acetone solvent system. In aqueous solvent systems such as pure water and 10% water solutions, Form B is the most stable of the polymorphic forms of 3- (4-amino-1-oxo-1,3-dihydro-isoindol-2-yl ) -piperidine-2,6-dione. However, it can become Form E in the presence of water. Desolvation experiments show that after heating at 175 ° C for five minutes, Form B typically becomes Form A.

When stored for a period of 85 days under two different temperature / relative humidity conditions (ambient temperature / 0% RH and 40 ° C / 93% RH), Form B does not convert into a different form.

In sum, Form B is a crystalline solid, hemihydrate, which has a DSC thermogram that exhibits endotherms at 146 and 268 ° C. Interconversion studies show that Form B is converted to Form E in aqueous solvent systems, and in other forms it is converted into acetone and other anhydrous systems.

C form

Form C can be obtained from evaporations, suspensions and slow cooling in acetone solvent systems. A representative XRPD pattern of this form is shown in Figure 12. The data is

characterized by peaks at 15.5 and 25 degrees 2θ.

Proton NMR in solution indicates that the 3- (4-amino-1-oxo-1,3-dihydro-isoindol-2-yl) -piperidine2,6-dione molecule is intact. Representative IR and Raman spectra are shown in Figures 13 and 14, respectively. The IR spectrum of Form C is characterized by peaks at 3466, 3373 and 3318 cm -1. The Raman spectrum of Form C is characterized by peaks at 3366, 3321, 1101 and 595 cm -1.

Representative thermal characteristics are represented for Form C in Figure 15. Form C loses 10.02% volatiles up to 175 ° C, indicating that it is a solvated material. Weight loss above 175 ° C is attributed to decomposition. The identification of volatiles in Form C can be carried out with TG-IR experiments. The representative IR spectrum captured after several minutes of heating, as depicted in Figure 13, when compared to a spectral library, shows that acetone is the one that best matches. Calculations made from TGA data show that Form C is a hemisolvate (0.497 moles of acetone). The DSC curve for Form C, shown in Figure 15, exhibits endotherms at 150 and 269 ° C. The endotherm at 150 ° C is attributed to the loss of the solvent, based on observations made during hot plate microscopy experiments. The endotherm at 269 ° C is attributed to the fusion, based on hot plate experiments.

Representative moisture sorption and desorption equilibrium data are shown in Figure 17. Form C does not exhibit a significant weight gain of 5 to 85% relative humidity, when equilibrium is obtained at each stage of relative humidity up to 85% relative humidity At 95% relative humidity, Form C experiences a significant weight loss of 6.03%. As the sample dries from 95% back to 5% relative humidity, the sample maintains the weight reached at the end of the adsorption phase at each stage up to 5% relative humidity. Form C is capable of becoming Form B when stored at 84% relative humidity for ten days.

Interconversion studies show that Form C is typically converted to Form A in a THF solvent system, and is typically converted to Form E into an aqueous solvent system. In an acetone solvent system, Form C is the most stable of 3- (4-amino-1-oxo-1,3-dihydro-isoindol-2-yl) -piperidine-2,6dione. Desolvation experiments performed on Form C show that after heating at 150 ° C for five minutes, Form C will typically become Form A.

In sum, Form C is a crystalline solid, hemisolvated, which has a DSC thermogram that exhibits endotherms at 150 and 269 ° C. Form C is not hygroscopic below 85% RH, but it can be converted to Form B at higher relative humidity.

Form D

Form D can be obtained from evaporation in acetonitrile solvent systems. A representative XRPD pattern of the shape is shown in Figure 18. The pattern is characterized by peaks at 27 and 28 degrees 2θ.

Proton NMR in solution indicates that the 3- (4-amino-1-oxo-1,3-dihydro-isoindol-2-yl) -piperidine2,6-dione molecule is intact. Representative IR and Raman spectra are shown in Figures 19 and 20, respectively. The IR spectrum of Form D is characterized by peaks at 3509, 2299 and 2256 cm -1. The Raman spectrum of Form D is characterized by peaks at 2943, 2889, 2297, 2260 and 1150 cm-1.

Representative thermal characteristics for Form D are plotted in Figure 21. Form D loses 6.75% volatiles up to 175 ° C, indicating that it is a solvated material. Weight loss above 175 ° C is attributed to decomposition. TG-IR experiments indicate that volatiles are water and acetonitrile. The calculations made from the TG data show that one mole of water is present in the sample. A representative DSC curve for Form D exhibits endotherms at 122 and 270 ° C. The endotherm at 122 ° C is attributed to the loss of volatiles based on observations made during hot plate microscopy experiments. The endotherm at 270 ° C is attributed to fusion based on hot plate experiments.

Representative sorption and moisture desorption data are plotted in Figure 22. Form D does not exhibit a significant weight gain of 5 to 95% relative humidity, when equilibrium is obtained at each stage of relative humidity. As the sample dries from 95% back to 5% relative humidity, the sample maintains its weight, such that at 5% relative humidity the form has typically gained only 0.39% by weight (0.012 mg) since The beginning to the end. Form D is capable of becoming Form B when stored at 84% relative humidity for ten days.

Interconversion studies show that Form D is capable of becoming Form A in a THF solvent system, Form E in an aqueous solvent system, and Form C in an acetone solvent system. Desolvation experiments performed on Form D show that after heating at 150 ° C for approximately five minutes, Form D will typically become Form A.

In sum, Form D is a crystalline solid, solvated with both water and acetonitrile, which has a DSC thermogram that exhibits endotherms at 122 and 270 ° C. Form D is weak or non-hygroscopic, but will typically become Form B when subjected to higher relative humidity.

E form

Form E can be obtained by suspending 3- (4-amino-1-oxo-1,3-dihydro-isoindol-2-yl) -piperidine-2,6-dione in water and by slow evaporation of 3- (4 -amino-1-oxo-1,3-dihydro-isoindol-2-yl) -piperidine-2,6-dione in a solvent system with a 9: 1 ratio of acetone: water. A representative XRPD pattern is shown in Figure 23.

The data are characterized by peaks at 20, 24.5 and 29 degrees 2θ.

Representative thermal characteristics of Form E are plotted in Figure 24. Form E typically loses 10.58% volatiles up to 125 ° C, indicating that it is a solvated material. A second weight loss of an additional 1.38% was observed between 125 ° C and 175 ° C. Weight loss above 175 ° C is attributed to decomposition. The Karl Fischer and TG-IR experiments support the conclusion that volatile weight loss in Form E is due to water. The representative DSC curve for Form E exhibits endotherms of 99, 161 and 269 ° C. Based on the observations made during hot plate microscopy experiments, the endotherms at 99 and 161 ° C are attributed to the loss of volatiles. The endotherm at 269 ° C is attributed to fusion based on hot plate experiments.

Representative sorption and moisture desorption data are plotted in Figure 25. Form E typically does not exhibit a significant weight change of 5 to 95% relative humidity when equilibrium is obtained at each stage of relative humidity. As the sample dries from 95% back to 5% relative humidity, the sample continues to maintain the weight, so that at 5% relative humidity the sample has lost only 0.0528% by weight from the beginning to the beginning. final.

Interconversion studies show that Form E can be converted into Form C in an acetone solvent system, and in Form G into a THF solvent system. In aqueous solvent systems, form E appears to be the most stable form. Desolvation experiments performed on Form E show that after heating at 125 ° C for five minutes, Form E can become Form B. After heating at 175 ° C for five minutes, Form B can become Form F.

When stored for a period of 85 days under two different temperature / relative humidity conditions (ambient temperature / 0% RH and 40 ° C / 93% RH), Form E typically does not convert to a different form. When stored for seven days at room temperature / 0% RH, Form E can become a new form, Form H.

F form

Form F can be obtained by complete dehydration of Form E. A representative XRPD pattern of the

Form F, shown in Figure 26, is characterized by peaks at 19, 19.5 and 25 degrees 2θ.

Representative thermal characteristics of Form F are shown in Figure 27. The representative DSC curve for Form F exhibits an endotherm at 269 ° C, directly preceded by two smaller endotherms indicative of a crystallized form of 3- (4-amino-1 -oxo-1,3-dihydro-isoindol-2-yl) -piperidine-2,6-dione. The DSC thermogram does not show any thermal event before melting, suggesting that it is a non-solvated material.

G form

Form G can be obtained by suspending forms B and E in THF. A representative XRPD pattern in this way, shown in Figure 28, is characterized by a peak at 23 degrees 2θ. Two other unique peaks of Form G appear at 21 and 24.5 degrees 2θ.

Representative thermal characteristics of Form G are plotted in Figure 29. A representative DSC curve for Form G exhibits an endotherm at 248 ° C, followed by a small, wide exotherm at 267 ° C. No thermal events are seen on the DSC thermogram at lower temperatures, suggesting that it is a non-solvated material.

H form

Form H can be obtained by storing Form E at room temperature and 0% RH for 7 days. A representative XRPD pattern is shown in Figure 30. The pattern is characterized by a peak at 15 degrees 2θ, and two other peaks at 26 and 31 degrees 2θ.

Representative thermal characteristics are shown in Figure 31. Form H loses approximately 1.67% of volatiles up to 150 ° C. Weight loss above 150 ° C is attributed to decomposition. Karl Fischer data shows that Form H typically contains 1.77% water (0.26 mol), suggesting that the weight loss seen in the TG is due to dehydration. The DSC thermogram shows a wide endotherm between 50 ° C and 125 ° C, which corresponds to the dehydration of Form H, and a narrow endotherm at 269 ° C, which is probably due to a fusion.

When suspended in water either with Form A or with B, after 14 days, Form H may become Form E. When suspended in THF, Form H may become Form A. When suspended in acetone, Form H can become Form C.

In sum, Form H is a crystalline, hydrated solid with 0.25 moles of water that has a DSC thermogram that exhibits an endotherm between 50 and 125 ° C, and an endotherm at 269 ° C.

Methods of use and pharmaceutical compositions

The polymorphs described herein exhibit physical characteristics that are beneficial for the manufacture, storage or use of drugs. All polymorphs described herein have utility as pharmaceutically active ingredients or intermediates thereof.

Methods for treating and preventing a wide variety of diseases and conditions using polymorphs of 3- (4-amino-1-oxo-1,3-dihydro-isoindol-2-yl) -piperidine-2,6- are described herein. diona In each of the methods, a therapeutic or prophylactically effective amount of the compound is administered to a patient in need of such treatment or prevention. Examples of such diseases and conditions include, but are not limited to, diseases associated with unwanted angiogenesis, cancer (eg, solid and blood-borne tumors), inflammatory diseases, autoimmune diseases and immune diseases. Examples of cancers and precancerous ailments include those described in US Pat. us. 6,281,230 and 5,635,517 to Muller et al. and in various US patent applications of Zeldis, including requests us. 10 / 411,649, filed on April 11, 2003 (Treatment of Myelodysplastic Syndrome); 10 / 438,213 filed on May 15, 2003 (Treatment of Various Types of Cancer); 10 / 411,656, filed on April 11, 2003 (Treatment of Myeloproliferative Diseases). Examples of other diseases and disorders that can be treated or prevented using the compositions of the invention are described in US Pat. us. 6,235,756 and

6,114,335 to D’Amato and other US patent applications. of Zeldis, which include 10 / 693,794, filed on October 23, 2003 (Treatment of Pain Syndrome) and 10 / 699,154, filed on October 30, 2003 (Treatment of Macular Degeneration).

Depending on the disease to be treated and the condition of the patient, the polymorphs of the invention can be administered orally, parenterally (e.g., intramuscularly, intraperitoneally, intravenously, ICV, intracisternal injection or infusion, subcutaneous injection or implantation), by inhalation, nasal, vaginal, rectal, sublingual or topical spray, and can be formulated, alone or together, in suitable unit dosage formulations containing excipients, adjuvants and non-toxic pharmaceutically acceptable carriers, appropriate for each route of administration. Because the individual polymorphs have different dissolution, stability and other properties, the optimal polymorph used in the treatment methods may depend on the route of administration. For example, forms that are readily soluble in aqueous solutions are preferably used to provide liquid dosage forms, while forms exhibiting high thermal stability may be preferred in the manufacture of solid dosage forms (e.g., tablets and tablets). capsules)

Although the physical characteristics of the polymorphs may, in some cases, affect their bioavailability, the amounts of the polymorphs that are therapeutically or prophylactically effective in the treatment of various diseases or ailments can easily be determined by experts accustomed to the pharmaceutical technique or medical For example, a polymorph is administered orally and in a single or divided daily dose, in an amount of 0.10 to 150 mg / day, or 5 to 25 mg / day or every other day in an amount of 0, 10 to 150 mg / day or 5 to 25 mg / day.

Simple pharmaceutical compositions and unit dosage forms are described herein which can be used in treatment and prevention methods, comprising one or more polymorphs of 3- (4-amino-1-oxo1,3-dihydro-isoindole-2- il) -piperidine-2,6-dione and optionally one or more excipients or diluents. Specific compositions and dosage forms are described in the various patents and patent applications cited herein. In one embodiment, a simple dosage form comprises a polymorph (eg, Form B) in an amount of 5, 10, 25 or 50 mg.

6. Examples 6.1. Polymorph screening

Polymorph screening was performed to generate the different solid forms of 3- (4-amino-1-oxo-1,3-dihydroisoindol-2-yl) -piperidine-2,6-dione as follows.

A heavy sample of 3- (4-amino-1-oxo-1,3-dihydro-isoindol-2-yl) -piperidine-2,6-dione (usually 10 mg) was treated with aliquots of the test solvent. The solvents were of reactive quality or HPLC quality. The aliquots were usually about 200 μl. Between additions, the mixture was usually stirred or sonicated. When the solids dissolved, which was judged by visual inspection, the estimated solubilities were calculated. The solubilities were estimated from these experiments based on the total solvent used to provide a solution. Actual solubilities may have been greater than those calculated due to the use of solvent aliquots too large or at a slow dissolution rate.

Samples were created generating solutions (usually 30 mg in 20 ml) at elevated temperatures, filtering, and allowing the solution to evaporate, either in an open vial (rapid hot evaporation) or in a vial covered with aluminum foil containing small holes (slow hot evaporation).

Suspension experiments were also performed. Usually about 25 mg of solid was placed in 3 or 5 ml of solvent. The samples were then placed in orbital shakers at room temperature or 40 ° C for 4-10 days.

Crystallizations were performed using various cooling methods. The solid was dissolved in a solvent at an elevated temperature (eg, 60 ° C), filtered quickly and allowed to cool to room temperature. Once at room temperature, samples that did not crystallize were taken to a refrigerator. The solids were removed by filtration or decantation and allowed to air dry. Rapid cooling was carried out by dissolving the solid in a solvent at an increased temperature (eg, 45-65 ° C) followed by cooling in an ice / acetone bath.

Hygroscopicity studies were performed by placing portions of each polymorph in a chamber at 84% relative humidity for a week.

Desolvation studies were carried out by heating each polymorph in an oven at 70 ° C for one week.

Interconversion experiments were carried out preparing suspensions containing two forms in a saturated solvent. The suspensions were stirred for 7-20 days at room temperature. Insoluble solids were recovered by filtration and analyzed using XRPD.

6.2. Preparation of polymorphic forms

Eight solid forms of 3- (4-amino-1-oxo-1,3-dihydro-isoindol-2-yl) -piperidine-2,6-dione were prepared as described below.

Form A was obtained by crystallization from various non-aqueous solvents, including 1-butanol, butyl acetate, ethanol, ethyl acetate, methanol, methyl ethyl ketone and tetrahydrofuran. Form B was also obtained by crystallization, from the hexane, toluene and water solvents. Form C was obtained from evaporations, suspensions and slow cooling in acetone solvent systems. Form D was obtained from evaporations in acetonitrile solvent systems. Form E was obtained, in the easiest way, by suspending 3- (4-amino-1-oxo-1,3-dihydro-isoindol-2-yl) -piperidine-2,6-dione in water. Form F was obtained by complete desolvation of Form E. It is found to be a non-solvated crystalline material that melts at 269 ° C. Form G was obtained by suspending Forms B and E in THF. Form H was obtained by subjecting Form E at room temperature and 0% RH for 7 days.

6.2.1. Synthesis of polymorphs B and E

Form B is the desired polymorph for the active pharmaceutical ingredient (API) of 3- (4 amino-1-oxo-1,3-dihydro-isoindol-2-il) -piperidine-2,6 -Diona. This form has been used in the formulation of the API in a pharmaceutical product for clinical studies. Three batches were produced as apparent mixtures of polymorphs in the non-micronized API of 3- (4-amino-1-oxo-1,3-dihydro-isoindol-2-yl) -piperidine-2,6-dione.

A development work was carried out to define a procedure that generated polymorph B from this mixture of polymorphs and could be implemented for strict polymorphic controls in the validation lots and the future manufacture of the 3- (4-amino API) -1-oxo-1,3-dihydro-isoindol-2-yl) -piperidine-2,6-dione. The characterization of the polymorphic forms produced during the work was carried out by XRPD, DSC, TGA and KF.

A procedure for the large-scale preparation of Form E was also developed. A material of polymorph E was prepared in order to carry out a comparison with the pharmaceutical product of polymorph B in a 3- (4 capsule dissolution test). -amino-1-oxo-1,3-dihydro-isoindol-2-yl) -piperidine-2,6-dione. 150 g of a mixture of polymorphs in 3 l of water were stirred at room temperature for 48 hours. The product was collected by filtration and dried at 25 ° C for 24 hours under vacuum. The XRPD, DSC, TGA, KF and HPLC analyzes confirmed that the isolated material was polymorph E.

In a preliminary work, it was shown that stirring a suspension of a mixture of 3- (4-amino-1-oxo1,3-dihydro-isoindol-2-yl) -piperidine-2,6-dione polymorphs with water at high Temperature (75 ° C) for an extended period of time converted this mixture of polymorphs exclusively into Form B. Several specific parameters were identified, including temperature, solvent volume and drying parameters (temperature and vacuum). XRPD, DSC, TGA, KF and HPLC analyzes were used to characterize all batches. After completing the optimization work, the optimized procedure was scaled up to 100-200 g on three API batches. Drying studies were carried out at 20 ° C, 30 ° C and 40 ° C, and 65 ° C, with a vacuum of 150 mm Hg. The results are shown in Tables 1-5.

The cooling and maintenance periods of a suspension of 3- (4-amino-1-oxo-1,3-dihydroisoindol-2-yl) -piperidine-2,6-dione were studied. Experimental laboratory data suggest that polymorph B appears to be forming first, and over time polymorph E equilibration occurs at ambient temperature conditions, thus generating a mixture of polymorphs B and E. This result supports the fact that Polymorph B appears to be a kinetic product, and that a prolonged processing time converts the material into polymorph E, resulting in a mixture of polymorphs B and E.

A laboratory procedure was developed to exclusively produce polymorph B of 3- (4-amino-1-oxo-1,3-dihydro-isoindol-2-yl) -piperidine-2,6-dione. The procedure includes a 10 volume aqueous suspension stirred at 75 ° C for 6-24 hours. The following preferred procedure parameters have been identified:

1. Hot suspension temperature of 70-75 ° C

5 2. Product filtration of 3- (4-amino-1-oxo-1,3-dihydro-isoindol-2-yl) -piperidine-2,6-dione at 65-75 ° C.

3.

Vacuum drying at 60-70 ° C is preferred for effective removal of unbound water in the wet paste of 3- (4-amino-1-oxo-1,3-dihydro-isoindole-2-yl) -piperidine- 2,6-dione.

Four.

The filtration step of 3- (4-amino-1-oxo-1,3-dihydro-isoindol-2-yl) -piperidine-2,6-dione can be a time-sensitive operation. The use of effective solid-liquid separation equipment is preferred.

10 5. Maintenance periods of 3- (4-amino-1-oxo-1,3-dihydro-isoindol-2-yl) -piperidine-2,6 dione paste at a KF higher than 5% may cause the kinetic balances of polymorph B to mixed polymorphs of E and B.

Drying up to KF <4.0% water was reached in 3 hours (30-70 ° C, 152 mm Hg). Polymorphs B and E were distinguished by water levels measured by KF and TGA. The polymorph B reference sample is API

15 micronized. In order to make an exact comparison by XRPD, the samples were gently ground before being sent for analysis. This increases the clarity of the polymorphic form identification. All samples were analyzed by XRPD, DSC, TGA, KF and HPLC.

Table 1: Preliminary studies

Quantity

Reaction conditions Analysis Results / conclusion

2 g

Water, ta, 48 h XRPD, DSC, TGA, KF Polymorph E

25 g

Water, ta, 48 h XRPD, DSC, TGA, KF Polymorph E

5 g

Water, 70-75 ° C, 24 h, then 24 h XRPD, DSC, TGA, KF Polymorph B

1 g

Acetone-Water 9: 1, evap. slow XRPD, DSC, TGA, KF Polymorph Mix

1 g

175 ºC 1 h in an oven XRPD, DSC, TGA, KF Polymorph A

0.5 g (polymorph A)

Water, ta, 24 h XRPD, DSC, TGA, KF Polymorph E

1 g of polymorph B

Water, ta, 48 h XRPD, DSC, TGA, KF Polymorph E

1 g of polymorph E

Water, 70-75 ° C, 24 h XRPD, DSC, TGA, KF Polymorph B

1 g

Heptane Suspension XRPD, DSC, TGA, KF Without changes

Table 2: Solvent Temperature, Time and Volume Optimization

Quantity

Amount of water (ml) Temp. (ºC) Time (h) Results / conclusion

10 g

fifty 75 6 Mixture

10 g

fifty 75 24 Polymorph B

10 g

100 70 6 Polymorph B

10 g

100 70 14 Polymorph B

10 g

100 70 twenty-one Polymorph B

10 g

100 75 6 Polymorph B

10 g

100 75 24 Polymorph B

10 g

100 75 6 Polymorph B

10 g

100 75 19 Polymorph B

10 g

100 75 14 Polymorph B

10 g

100 75 24 Polymorph B

5 g

100 75 18 Polymorph B

Quantity

Amount of water (ml) Temp. (ºC) Time (h) Results / conclusion

10 g

fifty 75 6 Mixture

10 g

fifty 75 24 Polymorph B

10 g

100 70 6 Polymorph B

10 g

100 70 14 Polymorph B

10 g

100 70 twenty-one Polymorph B

10 g

100 75 6 Polymorph B

10 g

100 75 24 Polymorph B

10 g

100 75 6 Polymorph B

10 g

100 75 19 Polymorph B

10 g

100 80 6 Polymorph B

10 g

100 80 twenty Polymorph B

10 g

200 Four. Five 6 Polymorph B + E

10 g

200 Four. Five 24 Polymorph E

10 g

200 60 48 Polymorph B

10 g

200 75 6 Mixture

10 g

200 75 24 Polymorph B

10 g

200 75 13 Polymorph B

10 g

200 75 24 Polymorph B

The optimum conditions were determined to be 10 volumes of solvent (H2O), 70-80 ° C for 6-24 hours.

Table 3: Maintenance Time

Quantity

Reaction conditions Maintenance Time (h) Temp. maintenance (ºC) Results / conclusion

5 g

Water, 70-75ºC, 24 h 24 23-25 Polymorph B

1 g of Polymorph B

Water, 70-75ºC, 24 h 48 23-25 Polymorph E

2 g

Water, 40 ml  16 23-25 Polymorph E

150 g

Water, 3.0 l  24 23-25 Polymorph E

150 g

Water, 3.0 l  48 23-25 Polymorph E

10 g

Water, 100 ml, 24 h, 75 ° C 18  23-25 Polymorph B

10 g

Water, 100 ml, 24 h, 75 ° C 18  40 Polymorph B

10 g

Water, 200 ml, 24 h, 75 ° C 14  -5 Mixture

10 g

Water, 200 ml, 24 h, 75 ° C 14  23-25 Polymorph E

10 g

Water, 200 ml, 24 h, 75 ° C 14  40 Mixture

10 g

Water, 100 ml, 24 h, 75 ° C twenty-one  23-25 Polymorph E

10 g

Water, 100 ml, 24 h, 75 ° C twenty-one  40 Mixture

10 g

Water, 100 ml, 14 h, 75 ° C 2 23-25 Mixture

The maintenance time gave mixed results, and it was determined that the material should be filtered at 60-65 ° C and the material washed with 0.5 volumes of hot water (50-60 ° C).

Table 4: Scale Increase Experiments

Quantity

Amount of water (l) Temp. (ºC) Time (h) Results / conclusion

100g

1.0  75 6 Polymorph B

100g

1.0  75 22 Polymorph B

100g

1.0  75 6 Polymorph B

100g

1.0  75 24 Polymorph B

100g

1.0  75 6 Polymorph B

100g

1.0  75 22 Polymorph B

Table 5: Drying Studies

Quantity

Drying Time (h) Temp. Drying (ºC) Vacuum (mm Hg) KF § (%) Results / conclusion

100g

0 - - 3,690 Polymorph B

100g

3 30 152 3,452 Polymorph B

100g

8 30 152 3,599 Polymorph B

100g

0 - - 3,917 Polymorph B

100g

5 40 152 3,482 Polymorph B

100g

22  40 152 3,516 Polymorph B

100g

3 40 152 3.67 Polymorph B

100g

22  40 152 3.55 Polymorph B

* Reaction conditions: Water 1 l, 75 ° C, 22-24 h; §: Average of 2 measures

Drying studies determined that the material should be dried at 35-40 ° C, 125-152 mm Hg for 3 to

22 h or until the water content reaches ≤ 4% by weight.

5 For a large-scale preparation of polymorph E (5222-152-B), a 5 l round bottom flask was charged with 3- (4-amino-1-oxo-1,3-dihydro-isoindole-2- il) -piperidine-2,6-dione (150 g, 0.579 mol) and water (3000 ml, 20 vol). The mixture was mechanically stirred at room temperature (23-25 ° C) for 48 h under a nitrogen atmosphere.

Samples were taken after 24 h and 48 h before filtering the sample and air drying in the filter for 1 h. The material was transferred to a drying tray and dried at room temperature (23-25 ° C) for 24 h. The analysis

10 KF on the dried material showed a water content of 11.9%. The material was submitted for analysis by XRPD, TGA, DSC and HPLC. The analyzes showed that the material was pure polymorph E.

For a large-scale preparation of polymorph B (5274-104), a 2-necked round bottom 3-neck flask was loaded with 3- (4-amino-1-oxo-1,3-dihydro-isoindole- 2-yl) -piperidine-2,6-dione (mixture of polymorphs, 100 g, 0.386 mol) and water (1000 ml, 10 vol). The mixture was heated at 75 ° C for approximately 30 minutes with stirring.

15 mechanics in a nitrogen atmosphere.

Samples were taken after 6 h and 24 h before allowing the mixture to cool to 60-65 ° C, filtered and the material washed with hot water (50-60 ° C) (50 ml, 0.5 vol). The material was transferred to a drying tray and dried at 30 ° C, 152 mm Hg for 8 h. KF analysis on the dried material showed a water content of 3.6%. After grinding the material, it was submitted for analysis by XRPD, TGA, DSC and HPLC. The analyzes showed that the material

20 was pure polymorph B. The results of the analyzes are shown in Figures 32-46.

6.3 X-ray powder diffraction measures

X-ray powder diffraction analyzes were carried out on a Shimadzu XRD-6000 X-ray powder diffractometer using Cu Ka radiation. The instrument is equipped with a thin focus X-ray tube. The tube voltage and amperage were set at 40 kV and 40 mA, respectively. The slits of divergence and dispersion are

25 set to 1 ° and the receiving slit was set at 0.15 mm. The diffracted radiation was detected by a scintillation detector of NaI. A continuous theta-dos theta scan was used at 3 ° / min (0.4 s / 0.02 ° stages) from 2.5 degrees 2θ to 40 degrees 2θ. A silicon pattern was analyzed each day to check the alignment of the instrument.

X-ray powder diffraction analyzes were also carried out using Ka del Cu radiation on an Inel XRG-3000 diffractometer equipped with a curved position sensitive detector. Data were collected in real time over a theta-dos theta interval of 120º at a resolution of 0.03º. The tube voltage and current were 40 kV and 30 mA, respectively. A silicon pattern was analyzed each day to check the alignment of the instrument. Only the region between 2.5 and 40 degrees 2θ is shown in the figures.

6.4. Thermal analysis

TG analyzes were performed on a TA Instruments TGA 2050 or 2950 instrument. The calibration standards were nickel and alumel. Approximately 5 mg of the sample was placed in a container, weighed accurately, and inserted into the TG oven. The samples were heated under nitrogen at a rate of 10 ° C / min, to a final temperature of 300 or 350 ° C.

DSC data were obtained on a TA 2920 instrument. The calibration standard was Indian. Samples of approximately 2-5 mg were placed in a DSC container and the weight was recorded accurately. Corrugated containers with a small hole were used for the analysis, and the samples were heated under nitrogen at a rate of 10 ° C / min, to a final temperature of 350 ° C.

Hot plate microscopy was carried out using a Kofler hot stage in a Leica microscope. The instrument was calibrated using USP standards.

For TG-IR experiments, a TA Instruments 2050 TGA interconnected with a Nicolet Fourier Fourier transform IR spectrophotometer was used, equipped with a Globar source, XT / KBr splitter, and deuterated triglycine sulfate (DTGS) detector ). The IR spectrometer was calibrated in wavelength with polystyrene on the day of use, while the TG was calibrated in temperature and weight biweekly, using indium for temperature calibration. A sample of approximately 10 g of 3- (4-amino-1-oxo-1,3-dihydro-isoindol2-yl) -piperidine-2,6-dione was weighed in an aluminum container and heated from 25 to 30 ºC up to 200 ºC at a speed of 20 ºC / min with a helium drain. IR spectra were obtained in series, each spectrum representing 32 co-added scans at a resolution of 4 cm -1. The spectra were collected with a repetition time of 17 seconds. TG / IR analysis data are presented as graphical representations of Gram-Schmidt and time-related IR spectra. Graphical representations of Gram-Schmidt show total IR intensity versus time; Hence, volatiles can be identified at each time point. They also show when volatiles are detected. From the graphical representations of Gram-Schmidt, time points were selected and the IR spectra of these time points are presented in the stacked related spectra. Each spectrum identifies the volatiles that are released at that point of time. The volatiles were identified from a search of the spectral library in the vapor phase of the RH Nicolet TGA. The matching results of the library are also presented to show the identified vapor.

6.5 Spectroscopy measurements

Raman spectra were acquired on a Nicolet Fourier Fourier transform Raman spectrometer using an excitation wavelength of 1064 nm and approximately 0.5 W of Nd: YAG laser energy. The spectra represent 128 to 256 co-added scans acquired at a resolution of 4 cm -1. The samples were prepared for analysis by placing the material in a sample container and positioning it on the spectrometer. The spectrometer was calibrated in wavelength using sulfur and cyclohexane at the time of use.

The average IR spectra were acquired on a Nicolet Fourier Fourier transform IR spectrophotometer equipped with a Globar source, XT / KBr splitter and a deuterated triglycine sulfate detector (DTGS). A Spectra-Tech, Inc. diffuse reflectance fixture was used for sampling, each spectrum representing 128 co-added scans at a spectral resolution of 4 cm-1. A set of background data was acquired with an alignment mirror in place. A single ray sample data set was then acquired. Subsequently, a log 1 / R spectrum (where R = reflectance) was acquired by relating the two sets of data against each other. The spectrophotometer was calibrated (wavelength) with polystyrene at the time of use.

6.6. Sorption / moisture desorption measures

The moisture sorption / desorption data was collected in a VTI SGA-100 moisture balance system. For the sorption isotherms, a sorption range of 5 to 95% relative humidity (RH) and a desorption range of 95 to 5% RH in increments of RH of 10% were used for the analysis. The sample was not dried before analysis. The equilibrium criteria used for the analysis were less than 0.0100 percent change in weight in 5 minutes with a maximum equilibration time of 3 hours if the weight criterion was not met. The data was not corrected for the initial moisture content of the samples.

6.7. NMR measurements of protons in solution

NMR spectra not previously reported were collected at SSCI, Inc., 30365 Kent Avenue, West Lafayette, Indiana. The 1H NMR spectra in the dissolution phase were acquired at room temperature on a Bruker model AM spectrometer. The 1H NMR spectrum represents 128 co-added transients collected with a pulse of 4 μs and a relaxation delay time of 5 seconds. The free induction decay (FID) was exponentially multiplied with a Lorentzian line widening factor of 0.1 Hz to improve the signal to noise ratio. The NMR spectrum was processed using GRAMS software, version 5.24. The samples were dissolved in dimethylsulfoxide-d6.

The scope of this invention can be understood with reference to the appended claims.

6.8. Intrinsic dissolution and solubility studies

Intrinsic dissolution experiments were performed on Form A (anhydrous), Form B (hemihydrate), and Form E (dihydrate) of 3- (4-amino-1-oxo-1,3-dihydro-isoindole-2- il) -piperidine-2,6-dione. Equilibrium solubility experiments were performed on Forms A and B. Aliquots were analyzed by ultraviolet-visible spectrometry, and the remaining solids of each experiment were analyzed by X-ray powder diffraction (XRPD).

6.8.1. Experimental part 6.8.1.1 Dissolution

The dissolution experiments were carried out in a VanKel VK6010-8 dissolution apparatus equipped with a VK650A heater / circulator. An intrinsic dissolution apparatus (Woods apparatus) was used. Samples were compressed at 1.5 metric tons for 1 min using the Woods apparatus in a hydraulic press, giving a sample area of 0.50 cm2. A dissolution medium consisting of 900 ml of HCl buffer, pH 1.8, with 1% sodium lauryl sulfate was used for each experiment. The medium was degassed by vacuum filtration through a 0.22 µm nylon filter disc and maintained at 37 ° C. The apparatus was rotated at 50 rpm for each experiment. Aliquots were filtered immediately using 0.2 µm syringe filters. In some cases, undissolved solids will

recovered and analyzed by X-ray powder diffraction (XRPD).

6.8.1.2. Solubility

Equilibrium solubility experiments were performed in a 100 ml three-necked round bottom flask immersed in a constant temperature oil bath at 25 ° C. A solid sample of 400-450 mg in 50 ml of dissolution medium (HCl buffer, pH 1.8, with 1% sodium lauryl sulfate) was stirred using a stir bar

mechanics. Aliquots were filtered using 0.2 μm syringe filters and immediately diluted 1 ml → 50 ml, then 5 ml → 25 ml, with dissolution medium in a glass container, a final dilution factor of 250.

6.8.1.3. UV-Vis spectrophotometry

Dissolution and solubility sample solutions were analyzed by a Beckman DU 640 single ray spectrophotometer. A quartz cuvette of 1,000 cm and an analysis wavelength of 228.40 nm was used. The detector was zeroed with a cuvette filled with dissolution medium.

6.8.1.4 X-ray powder diffraction

XRPD analyzes were performed on a Shimadzu XRD-6000 X-ray powder diffractometer using the

Kα radiation of Cu. The instrument is equipped with a thin focus X-ray tube. Energy and amperage was set

of the tube at 40 kV and 40 mA, respectively. The divergence and dispersion slits were set at 1 ° and the receiving slit was set at 0.15 mm. The diffracted radiation was detected by a scintillation detector of NaI. A continuous theta-dos theta scan was used at 3 ° / min (0.4 s / 0.02 ° stages) from 2.5 to 40 degrees 2θ. A silicon pattern was analyzed each day to check the alignment of the instrument. The samples were packaged in an aluminum container with silicon insert.

6.8.2. Results

The results of these intrinsic solubility studies are summarized in Table 6. Both the solubility and dissolution experiments were performed in an HCl buffer medium, pH 1.8, containing 1% sodium lauryl sulfate. Form A was found to be unstable in the medium, becoming Form E. The solubilities of Forms A, B and E were estimated to be 6.2, 5.8, and 4.7 mg / ml, respectively. The dissolution rates of Forms A, B and E were estimated to be 0.35, 0.34 and 0.23 mg / ml, respectively.

6.8.2.1 Development of the UV-Vis spectrophotometry method

A UV-Vis scan of the dissolution medium (set as blank with an empty cuvette) was made to identify any interfering peaks. A small peak at 225 nm was present, as shown in Figure 47.

Solutions of 3- (4-amino-1-oxo-1,3-dihydro-isoindol-2-yl) -piperidine-2,6-dione at varying concentrations were analyzed by UV-Vis spectrophotometry. A preliminary scan of a 1.0 mg / ml solution was made, with the instrument taken to the blank with dissolution medium. The solution was slightly absorbent and noisy between 200 280 nm, making dilution necessary.

A 0.04 mg / ml solution of 3- (4-amino-1-oxo-1,3-dihydro-isoindol-2-yl) -piperidine2,6-dione was then scanned between 200-300 nm . The graphic representation was still noisy between 200 and 230 nm, as shown in Figure 48. The sample was further diluted to 0.08 mg / ml. A wavelength scan between 200-350 nm for this sample showed a peak at 228.4 nm, without interference, as shown in Figure 49. Therefore, a wavelength of 228.4 nm was chosen for the analysis of the solubility and dissolution of the samples.

5 A six-point calibration curve was generated with standards of the following concentrations: 0.001 mg / ml, 0.002 mg / ml, 0.005 mg / ml, 0.010 mg / ml, 0.015 mg / ml and 0.020 mg / ml (Notepad 569-90). A linearity coefficient of R2 = 0.9999 was obtained, as shown in Figure 50.

6.8.2.2 Solubility

A sample consisting of 449.4 mg of Form A was suspended in dissolution medium. The

10 particle size. Aliquots were taken at 7, 15, 30, 60, 90 and 150 min. The concentration reached 6.0 mg / ml at the first time point. The highest concentration reached was 6.2 mg / ml, at 30 min. From that point the concentrations decreased, reaching 4.7 mg / ml at 150 min as shown in Figure 51. The solids that remained at the final time point were analyzed by XRPD and found to be Form E, as was shown in Table 7. You cannot see peaks attributed to Form A in the pattern. Since the concentration is not

15 stabilized at 4.7 mg / ml, the solubility of Form E may be lower than that.

A sample consisting of 401.4 mg of Form B was suspended in dissolution medium. The particle size was not controlled. Aliquots were taken at 7, 15, 30, 60, 960, 180, 420 and 650 min. Form B dissolved much more slowly than Form A, reaching 3.3 mg / ml in 90 min. The concentration stabilized at 5.6 - 5.7 mg / ml at the three final time points as seen in Figure 52. It was shown that the solids that remained were Form B

20 as seen in Table 7, suggesting that Form B has good water stability.

A summary of the solubilities is given in Table 6. The amounts dissolved at each time point are shown in Tables 8 and 9.

Table 6: Summary of Results

Shape

Solubility Intrinsic dissolution # 1 Intrinsic Dissolution # 2 Average intrinsic dissolution rate

Form A

6.2 mg / ml 0.35  0.22a  0.29 th

Form B

5.8 mg / ml 0.35  0.32 0.34

E form

4.7 mg / ml 0.21  0.25 0.23

to. The dissolution experiment of Form A # 2 may have become Form E on the surface of the disk, skewing the average speed to lower.

Table 7: Experimental Details Table 8: Solubility of Form A

Experiment

Final Form

Pressed Form A

TO

Pressed Form B

B

Solubility of Form A

AND

Solubility of Form B

B

Dissolution of Form A

-

Dissolution of Form A

TO

Dissolution of Form B

-

Dissolution of Form B

B

Dissolution of Form E

AND

Dissolution of Form E

-

Time point (min)

Concentration (mg / ml)

7

6.00

fifteen

 6.11

30

 6.16

60

 6.10

90

 5.46

150

 4.73

Table 9: Solubility of Form B

Time point (min)

Concentration (mg / ml)

7

1.63

fifteen

 2.14

30

 2.33

60

 2.94

90

 3.34

180

 5.67

420

 5.76

650

 5.61

6.8.2.3 Intrinsic dissolution

Approximately 200 mg of each of Forms A and B were compressed into discs in the Woods apparatus

5 using 2 metric tons of pressure. Subsequently, the samples were scraped, ground gently and analyzed by XRPD. The study showed that compression and grinding does not cause a change of form in any of the cases. (See Table 7).

Two preliminary dissolution executions were carried out. The discs were fractured to some extent in both experiments, compromising the requirement of constant surface area.

10 The first intrinsic dissolution experiment that strictly followed the USP chapter on intrinsic dissolution used approximately 150 mg of each of Forms A and B. Seven aliquots were taken, starting at 5 min and ending at 90 min, to maintain the conditions sedimentation The experiment resulted in linear dissolution profiles, at a rate of 0.35 mg per cm2 per minute for both forms. The experiment of Form E was done later under the same conditions, and added to the graph for

15 comparison. (See Figure 53). The dissolution rate of Form E was 0.21 mg per cm2 per minute, significantly lower than the dissolution rate of Forms A and B. This is in line with expectations based on solubility data. The crystalline form of the solids that remained did not change in any case.

The second experiment used approximately 250 mg of each of Forms A and B. The experiment of

Form E (135 mg) was made later and added to the chart for comparison. (See Figure 54). Nine aliquots were taken, starting at 5 min and ending at 150 min. The dissolution rates were 0.22, 0.32 and 0.25 mg per cm2 per minute, respectively, for Forms A, B and E. The dissolution rate for Form A in this experiment was low, while the Speeds for Forms B and E were similar to those found in the first experiment. It is believed that, in this case, a thin layer of the Form A disk may have

25 become Form E after exposure to water. This is supported by evidence of the rapid conversion of Form A into Form E in the solubility experiment. The diffraction pattern of undissolved solids does not indicate a change in shape. However, the thickness of the sample disc is not exposed to water. Therefore, it is believed that the actual intrinsic dissolution rate of Form A is close to 0.35 mg per cm2 per minute. An insufficient amount of Form A was available to repeat the experiment.

30 A summary of the intrinsic dissolution rates is given in Table 6. The amounts dissolved at each time point are summarized in Tables 10 and 11.

Table 10: Results of the Intrinsic Dissolution Experiment No. 1

Time point

Form A to Form B a Form E a

5 min

5.76 10.80 b  2.70

10 minutes

7.73 6.85 4.13

20 min

11.31 10.25 6.96

30 min

15.59 14.35 9.60

45 min

21.98 20.57 12.57

60 min

27.11 25.70 15.16

90 min

34.17 34.34 20.82

to. The results are reported as Cumulative Dissolved Amount per Unit Area (mg / cm2) b. This data point is not included in the graph, since the value is higher than the next two data points.

Table 11: Results of the Intrinsic Dissolution Experiment No. 2

Time point

Form A to Form B a Form E a

5 min

4.50 5.04 3.06

10 minutes

5.22 6.12 4.31

20 min

7.54 7.73 11.40

30 min

11.46 12.72 11.93

45 min

15.01 17.33 14.72

60 min

18.38 21.93 18.52

90 min

24.38 31.64 26.24

120 min

30.35 41.31 33.56

150 min

35.26 49.54 40.82

to. The results are reported as Cumulative Dissolved Amount per Area Unit (mg / cm2)

6.9. Analysis of polymorph mixtures

This invention encompasses mixtures of different polymorphs. For example, an X-ray diffraction analysis of a

5 production sample gave a pattern that contained two small peaks seen at 12.6º and 25.8º 2θ in addition to the representatives of Form B. In order to determine the composition of that sample, the following steps were performed:

1) Pairing of the new production pattern with known forms together with common pharmaceutical excipients and contaminants;

10 2) Cluster analysis of the additional peaks to identify if any unknown phase is mixed with the original Form B;

3) Harmonic analysis of the additional peaks to identify if any preferred orientation may be present or if a change in the crystalline habit may have occurred; Y

4) Indexing of unit cells for both Form B and the new production sample to identify any possible crystallographic relationships.

Based on these tests, which can be adapted for the analysis of any mixture of polymorphs, it was determined that the sample contained a mixture of polymorphic forms B and E.

6.10. Dosage form

Table 12 illustrates a batch formulation and a single dosage formulation for a single dose unit Pregelatinized corn starch components (SPRESS B-850) and polymorphic form of 3- (4-amino-1-oxo1,3- dihydro-isoindol-2-yl) -piperidine-2,6-dione are passed through a sieve (in this case, a 710 sieve

25 mg in a polymorphic form of 3- (4-amino-1-oxo-1,3-dihydro-isoindol-2-yl) -piperidine-2,6-dione.

Table 12: Formulation for a 25 mg capsule

Material

Weight percentage Amount (mg / tablet) Quantity (kg / lot)

Polymorphic form of 3- (4-amino-1-oxo1,3-dihydro-isoindol-2-yl) -piperidine-2,6dione

40.0%  25 mg 16.80 kg

Pregelatinized corn starch, NF

59.5% 37.2 mg 24.99 kg

Magnesium stearate

0.5% 0.31 mg 0.21 kg

Total

100.0% 62.5 mg 42.00 kg

μm) and then loaded into a Broadcast Mixer with a baffle insert and mixed during

approximately 15 minutes Magnesium stearate is passed through a sieve (in this case,

a sieve of 210 μm) and added to the Diffusion Mixer. Then, the mixture is encapsulated in capsules using

a Dosator type capsule filling machine.

Claims (11)

1. A process for preparing 3- (4-amino-1-oxo-1,3-dihydro-isoindol-2-yl) -piperidine-2,6-dione hemihydrate crystalline, which comprises the stages of: suspend 3- (4-amino-1-oxo-1,3-dihydro-isoindol-2-yl) -piperidine-2,6-dione in water at a temperature of 70 ° C at 80 ° C for 6 to 24 hours; filter 3- (4-amino-1-oxo-1,3-dihydro-isoindol-2-yl) -piperidine-2,6-dione; Y Dry 3- (4-amino-1-oxo-1,3-dihydro-isoindol-2-yl) -piperidine-2,6-dione in vacuo.

2.

The process of claim 1, wherein the suspension is carried out with 10 volumes of water.

3.

The method of claim 1 or 2, wherein the suspension is carried out at a temperature of 70 ° C to 75 ° C.

Four.

The method of any one of claims 1 to 3, wherein the filtration is carried out at a temperature from 60 ° C to 65 ° C or at a temperature of 65 ° C to 75 ° C.

5.

The method of any one of claims 1 to 4, wherein the drying is carried out at a temperature from 30 ° C to 70 ° C.

6.

The process of claim 5, wherein drying is carried out at a temperature of 35 ° C to 40 ° C

or from 60 ° C to 70 ° C.

7.

The process of any one of claims 1 to 6, wherein drying is carried out under vacuum from 125 mm Hg to 152 mm Hg.

8.

A process for preparing 3- (4-amino-1-oxo-1,3-dihydro-isoindol-2-yl) -piperidine-2,6-dione dihydrate crystalline, which comprises the stages of: suspend 3- (4-amino-1-oxo-1,3-dihydro-isoindol-2-yl) -piperidine-2,6-dione in water at room temperature;

filter 3- (4-amino-1-oxo-1,3-dihydro-isoindol-2-yl) -piperidine-2,6-dione; Y Dry 3- (4-amino-1-oxo-1,3-dihydro-isoindol-2-yl) -piperidine-2,6-dione.

9.

The method of claim 8, wherein the suspension is carried out with 20 volumes of water.

10.

The method of claim 8 or 9, wherein the suspension is carried out at a temperature of 23 ° C to 25 ° C.

eleven.

The method of any one of claims 8 to 10, wherein the drying is carried out at a temperature of 23 ° C to 25 ° C.